13232
J. Phys. Chem. C 2007, 111, 13232-13237
Nanoporous S-Layer Protein Lattices. A Biological Ion Gate with Calcium Selectivity Sofia Sotiropoulou,*,† Sonny S. Mark,†,‡ Esther R. Angert,§ and Carl A. Batt† Departments of Food Science and Microbiology, Cornell UniVersity, Ithaca, New York 14853 ReceiVed: March 16, 2007; In Final Form: June 12, 2007
S-layer protein lattice fragments isolated from the bacterium Deinococcus radiodurans were adsorbed onto chemically modified silicon substrates to produce nanoporous membranes containing uniformly sized pores with dimensions between 2 and 7 nm. The gating characteristics of the nanoporous membranes toward various ionic species were evaluated using electrochemical impedance spectroscopy. Our results reveal that the immobilized S-layers undergo a strong interaction with cations, especially Ca2+ ions. We observed for the first time ionic currents flowing through the nanoporous regions of the protein, pointing to an ion transport mechanism of gating. Our observations suggest that ion transport is mainly due to the presence of an electrical gradient inside the pores. The origin of the observed selectivity is discussed.
Introduction Nanoporous materials have been under intense investigation during the past decade.1,2 Initial interest in such systems stemmed from their crucial role in physiological processes3 such as ion channel conductance, nerve impulse propagation, etc. However, it has become evident that nanopores can also have applications in the design of biochemical separation systems4 and novel types of biosensors with single-molecule detection capabilities.5,6 The operational principle behind nanoporesensing elements lies in the blockage or rectification of the ionic current passing through the pore:7 when a small ion or molecule displaces electrolyte solution or water residing inside the pore, causing a transient change of aperture with resistance occurs with a consequent drop in the transmembrane voltage. The detection of ions and small molecules, as well as biomacromolecules, using nanopores has been demonstrated8,9 with sensitivities that can reach down to a single molecule.10 In most cases, the fabrication of nanoporous systems involves either the assembly of naturally occurring biomacromolecular structures (e.g., R-hemolysin pores,11 gramicidin, or porin proteins) or the deterministic fabrication of synthetic analogues using conventional lithographic processes (e.g., UV photolithography or electron-beam lithography). Although the former systems are characterized by high specificity, they are usually comprised of labile protein molecules that are held within fragile lipid bilayers. Ultimately, this renders such biologically based systems highly sensitive to external perturbations and also provides little control over pore morphology and distribution. Synthetic nanoporous systems, on the other hand, can be precisely manufactured to dimensions as small as 10 nm12,13 and are mechanically stable over a broad range of physical and chemical conditions. However, their fabrication often requires complicated, costly, and time-consuming multistep procedures. An attractive alternative to the lithographic fabrication of synthetic nanopores is the use of bacterial S-layer proteins. S-layer monomers are characterized by their exceptional ability to self-assemble in vivo into two-dimensional (2-D) crystalline * To whom correspondence should be addressed. E-mail: ss549@ cornell.edu. Phone: (607) 255-7902. Fax: (607) 255-8741. † Department of Food Science. ‡ Present address: Department of Pathology & Laboratory Medicine, 7.103 Founders Pavilion, Hospital of the University of Pennsylvania, University of Pennsylvania Medical Center, 3400 Spruce St., Philadelphia, PA 19104-4283. § Department of Microbiology.
arrays with oblique (p2), square (p4), or hexagonal (p3, p6) symmetries. The resulting monolayer structures are highly repetitive, nanoporous lattices of uniform morphology featuring well-defined pore sizes.14 The lattice constant of microbial S-layer protein sheets can range from 3 to 30 nm. The pore diameter can be exceptionally small and is typically in the range of only 2-6 nmsdimensions that are currently difficult to obtain even with sophisticated electron-beam-based lithographic techniques. Thus, a series of nanobiotechnological applications based on S-layer proteins have been envisaged. Indeed, the fabrication of nanoparticle arrays of well-defined lattices15,16 and the design of scaffolds for biological molecules,17 ion carriers, or poreforming proteins18,19 have been successfully reported. One of the initially hypothesized functions of the S-layer protein arrays is that of the molecular sieve and ion trap.17 Nevertheless, to date no detailed reports exist regarding their ion transport properties, and very few discussions have examined the potential use of S-layer membranes as nanopores for ultrasensitive sensor applications.20 Here we examine the iongating properties of an S-layer membrane array isolated from Deinococcus radiodurans with the aim of providing a biologically based alternative to current nanopore technologies. Experimental Section Materials. Si(100) wafer substrates (475-575 µm thickness, p-type/boron-doped, 1-5 Ω cm resistivity) with a ∼2 nm native oxide layer were purchased from Silicon Quest International (Santa Clara, CA). Dimethyldichlorosilane (DDS) was purchased from Gelest Co. (Milwaukee, WI). Citrate-capped gold nanoparticles (Au NPs; 5 nm nominal diameter, 80 nM in water) were purchased from Sigma Chemical Co. (St. Louis, MO). Unless stated otherwise, all other reagents (ACS grade or better) were purchased from either Aldrich Chemical Co. (Milwaukee, WI) or Sigma Chemical Co. and used as received. All aqueous solutions were prepared in reagent-grade water (DI H2O) (18.2 MΩ resistivity). Microscopy Characterization. Scanning electron microscopy (SEM) was carried out using a Zeiss Ultra scanning electron microscope equipped with a GEMINI field emission column. Atomic force microscopy (AFM) images were obtained under dry conditions using the PicoPlus AFM system from Molecular Imaging (Tempe, AZ). The probe tips used were ultrasharp silicon cantilevers with a typical resonance frequency of 155 kHz and a nominal force constant of 1.75 N m-1 (MikroMasch USA, Oregon).
10.1021/jp072132l CCC: $37.00 © 2007 American Chemical Society Published on Web 08/11/2007
Nanoporous S-Layer Protein Lattices
J. Phys. Chem. C, Vol. 111, No. 35, 2007 13233
Figure 1. D. radiodurans S-layer protein. (a) Schematic illustration (top-down view) of the morphological structure and hexagonal (p6) symmetry of the HPI S-layer. (b) Schematic illustration (side view) of the HPI S-layer fragments immobilized on the DDS-Si surface. (c) Three-dimensional surface view of the HPI S-layer intracellular face, as imaged by AFM using a carbon-nanotube-modified probe tip.
Figure 2. Electrochemical impedance characterization of the HPI S-layer bioelectronic device. (a) Schematic illustration of the semiconductorprotein interface and corresponding equivalent circuit used to model the impedance spectra. Rs, Cdl, Rct, and W represent the solution resistance, the double-layer capacitance, the charge-transfer resistance, and the Warburg impedance (diffusion resistance), respectively. (b, c) Characteristic Nyquist and Bode plots, obtained for DDS-derivatized silicon surfaces with immobilized native D. radiodurans (HPI) S-layer fragments. The spectra were acquired in DI H2O and were recorded over the frequency range 100 kHz to 1 Hz at 10 mV ac voltage and 0.0 mV dc voltage.
Electrochemical Measurements. Electrochemical impedance spectroscopy (EIS) and amperometry measurements were performed with a Gamry FAS2 Femtostat system using a standard three-electrode electrochemical cell setup. A Pt wire
was employed as a counter electrode (1.5 mm diameter, obtained from Aldrich Chemical Co.), and a Ag wire was used as a quasireference electrode (0.25 mm diameter, obtained from Aldrich Chemical Co.). The total cell volume was 150 µL, and the
13234 J. Phys. Chem. C, Vol. 111, No. 35, 2007 working surface area was 0.05 cm2. The sign convention of the potentiostat for all electrical current measurements is as follows: Positive values indicate reductive (cathodic) currents, and negative values indicate oxidative (anodic) ones. All impedance measurements were performed by applying a 10 mV ac voltage and a dc voltage at 0.0 mV over the 100 kHz to 1 Hz frequency range. The data are represented as either a Nyquist plot of the Zim part versus the Zreal part of the impedance or Bode plots (total impedance Z versus frequency and phase φ versus frequency). The interfacial capacitances were obtained by fitting the complex plane spectra (Nyquist plot) to an equivalent Randles circuit using the Echem Analyst software program (Gamry). Cell Culture Conditions and Purification of S-Layer Proteins. Growth of D. radiodurans strain Sark I and extraction of HPI S-layer protein fragments were performed as described previously. S-Layer Protein Adsorption to Chemically Modified Silicon Substrates. Si wafer substrates were cut into smaller pieces (∼22 × 26 mm) and rinsed in acetone, 2-propanol, and DI H2O under sonication. The Si chips were then exposed for 1 min to an air plasma generated in a Harrick Scientific benchtop plasma cleaner operated at high power (29.6 W). Chemical modification of the Si chips was carried out in the gas phase by placing the substrates inside a tightly sealed centrifuge tube containing 10 µL of saturated DDS vapor for 1 h at room temperature. This silanization procedure leads to a highly hydrophobic methylated surface. For the noncovalent immobilization of the S-layer proteins onto the DDS-modified Si chips, 10 µL of protein solution (50 µg mL-1 in DI H2O) was drop-cast onto the substrates and allowed to react for 1 h at room temperature. Subsequently, the chips were rinsed three times by immersion in DI H2O. This protocol leads to the efficient adsorption of large fragments of the HPI S-layer, as confirmed by AFM (Figure S2 in the Supporting Information). Adsorption of Gold Nanoparticles to S-Layer Proteins. To prevent the passage of ions through the vertex regions of the HPI S-layer, Au NPs were adsorbed to the surface of the protein. In the procedure, 50 µL of citrate-stabilized Au NPs in DI H2O was drop-cast onto the DDS-modified silicon chips immediately after the protein adsorption step. After 45 min, NaCl was added to the solution to a final concentration of 25 mM, and nanoparticle adsorption was continued for another 15 min. The silicon chips were then gently rinsed with DI H2O to remove any loosely bound nanoparticles. Au NP adsorption to the S-layer proteins was confirmed by SEM imaging. Results and Discussion Electrochemical Impedance Characterization of the SLayer Bioelectronic Device. The D. radiodurans S-layer, also known as the hexagonally packed intermediate (HPI) layer, consists of six identical protein monomers (each 98 kDa) that enclose a central pore with a diameter of ∼4 nm. The monomers, in turn, are surrounded by six identical vertex regions of ∼7 nm diameter (Figure 1a). Given its nanometric lattice dimensions and its robust and stable nature, the HPI S-layer is of particular interest for the development of bioelectronic devices. The fabrication of the HPI S-layer bioelectronic device was performed in two facile steps according to a previously optimized procedure:16 The first step involves the silanization of the silicon surface to render it hydrophobic, followed by direct chemisorption of the S-layer membrane fragments (Figure 1b). The AFM images of the as-immobilized protein sheets revealed a relatively smooth topography, which is attributed to the outer
Sotiropoulou et al.
Figure 3. Sensitivity of the S-layer biolectronic device to four chloride cations in DI H2O. Sensitivity was measured as capacitance per molar concentration, ∆C/[M], pF‚M-1. The inset is a schematic depiction of a positive field effect on a p-type semiconductor.
hydrophilic face of the S-layer protein. In contrast, the hydrophobic inner face usually appears more rugged (Figure 1c).15 The semiconductor-protein interface gives rise to a frequencydependent variation of the electrical response upon biomolecular recognition events,21 which can be monitored using EIS.22 In particular, the interaction of a protein membrane with ions is expected to induce changes in the transmembrane conductance, causing in turn changes in the capacitance of the double layer. Therefore, in this study, only capacitance values were extrapolated from the impedance spectra and used for further evaluation. The Nyquist plots of the native D. radiodurans (HPI) S-layer fragments in DI H2O consist only of semicircles (Figure 2b), indicating that the ionic processes taking place at the electrical double layer23 are in fact governing the behavior of the system. The occurrence of diffusion-limiting processes in the bulk solution or a pronounced iR drop would result in a straight line in the Nyquist plot. In the Nyquist plot (Figure 2b), the arc of the high-frequency semicircle (Figure 2b, blue fitted line) reflects a kinetically driven process and purely capacitive behavior; thus, we assigned this to the Helmholtz plane part of the double layer (Helmholtz capacitance Cdl,H ) A0/δ), which is very pronounced in highly doped semiconductor surfaces such as the silicon substrates used throughout this study.21 The Helmholtz capacitance was found to govern the response of the system. The low-frequency semicircle (Figure 2b, red fitted line) is a suppressed arc characteristic of a diffusion-controlled process.2 In solutions of increasing ionic strength (10-100 mM buffer or ion solutions), the low-frequency semicircle became further suppressed, revealing an inverse ionic strength dependence, and was thus assigned to the Gouy-Chapman diffusion layer (Gouy-Chapman capacitance Cdl,GC ) 0/LD). Although the Nyquist plot identified clearly the behavior of the system, a more practical representation of nonfaradic impedance spectra, commonly used when mainly capacitance values are extrapolated, is the Bode plot24 (Figure 2c). Thus, the Bode representation will be used henceforth in this paper. Ionic-Gating Characteristics of the D. radiodurans SLayer. The ion-gating characteristics of the HPI S-layer nanopores were evaluated by simultaneous recording of the impedance spectra (Figure 3) and monitoring of the open circuit potential of the S-layer membrane. Four chloride cations (M+Cl-, M ) Na+, K+, Ca2+, or Mg2+) and three sodium anions (Na+X-, X ) NO3- or SO42-; citrate-3- data not given) of
Nanoporous S-Layer Protein Lattices
J. Phys. Chem. C, Vol. 111, No. 35, 2007 13235
Figure 4. Calcium response of the HPI S-layer bioelectronic device. (a) Schematic representation of the possible mechanism of the calcium response of the S-layer device. (b) Bode plots of the S-layer device to DI H2O (9) and 10-6 (*), 10-5 (]), 10-4 (O), 10-3 (4), and 10-2 (0) M Ca2+ and corresponding response curve (inset). (c) Ionic current recorded upon addition of Ca2+ aliquots. The dc voltage was 50 mV vs the Ag quasireference electrode.
varying charge and lipophilic properties were examined in the concentration range 10-6 to 10-2 M. In all cases, the open circuit potential becomes more positive for increasing concentrations, indicating preferential adsorption of cationic species to the surface. This behavior correlates well with negatively charged synthetic nanopores that have been shown to display a similar cationic response.25 Furthermore, capacitance values decreased with increasing cation concentration (see also Figure S3 in the Supporting Information). This also correlates well with the effect of a positive (cationic) field on the p-doped Si surface that causes the valence and conduction bands to bend further, thus inducing the observed decrease in the interfacial capacitance (Figure 3, inset).26,27 Among the cations examined, the highest capacitive response was measured for Ca2+ (75.5 pF‚M-1). Sensitivity decreased in the following order: K+ (showing a sensitivity value of 25 pF‚M-1), followed by Mg2+ (11 pF‚M-1) and finally by Na+ (where practically no response was detected). The calcium response was significantly higher compared to that of the other cations examined. The selectivity series observed could not be directly correlated with the ionic charge, radius, or Hofmeister lipophilicity alone. These characteristics thus prompted us to further investigate the origins of the calcium response. Origin of the Calcium Response of the HPI S-Layer Bioelectronic Device. The S-layer bioelectronic device was found to have a selective response to Ca2+ ions. The response was measured as the change in capacitance upon addition of aliquots of calcium in DI H2O. The linear range of the response was between 10-4 and 10-2 M, with a sensitivity of 75.5 pF‚M-1. The calcium response was drastically affected by the ionic strength of the solution. Measurements in solutions of increasing ionic strength (10-100 mN) showed that the response was gradually masked, and in buffer solutions of 0.1 N ionic strength the response decreased by almost 60%. This clearly points to
electrostatic forces acting as the driving force for the ionic response, rather than the existence of affinity sites on the protein surface. Furthermore, the stepwise addition of Ca2+ aliquots to the electrochemical cell was found to cause the generation of an amperometric current. Each Ca2+ addition resulted in a stepwise increase in current recorded, as seen in Figure 4c. The current detected was cathodic, which corresponds to reductive currents or to a flux of cations toward the working electrode surface. For the concentration range 10-2 to 10-3M Ca2+, the total ionic response was 5 nA. These observations point to the possible transport of calcium ions occurring through the pore regions of the S-layer protein lattice structure. To test this hypothesis, we examined the calcium response of an HPI S-layer device whose vertex pore regions were occluded with Au nanoparticles. We have previously shown that Au nanoparticles (d ) 5 nm) mainly reside at the vertex regions of the HPI protein (Figure 5a),16 and this also allowed us to test the specific contribution of each pore region to the ionic response. Under these vertex-blocked conditions, no response to Ca2+ was detected even at 10-2 M concentrations. Moreover, no capacitance changes were found to occur (Figure 5b), nor could any detectable amperometric signal be recorded (Figure 5c). This verified the hypothesis that calcium response is occurring via a transport mechanism of ions mainly through the vertex regions of the S-layer protein. The amplitude of the calcium-induced amperometric signal of the vertex-free S-layer is higher than that reported for single, cylindrical-shaped nanopores, which typically lie in the picoampere range.28 This may be attributed to signal amplification that occurs through the array of vertex regions of the membrane, but may also be due to the effect of current rectification caused by electrical asymmetry (i.e., charge gradients occurring at the S-layer surface). The latter scenario cannot be ruled out, since
13236 J. Phys. Chem. C, Vol. 111, No. 35, 2007
Sotiropoulou et al.
Figure 5. Blocking of the vertex regions of the S-layer protein membrane blocks the ionic current transport of calcium ions through the pores of the protein. (a) Schematic representation of the Au NP S-layer device. (b) Bode plots of the blocked-S-layer device to DI H2O (9) and 10-6 (*), 10-5 (]), 10-4 (O), 10-3 (4), and 10-2 (0) M Ca2+ and corresponding response curve (inset). (c) Ionic current of the blocked-S-layer device recorded upon addition of Ca2+ aliquots. The dc voltage was 50 mV vs the Ag quasi-reference electrode.
there exists the possibility that the structural asymmetry of the HPI S-layer (due to inherent topological differences between its intracellular and extracellular sides) is accompanied by the presence of higher amounts of negative charge localized at the inner (i.e., intracellular) face, creating an increasingly negative potential drop toward the surface of the silicon. The role of electrical gradients in the ion transport was examined by recording the calcium response at pH 6.7, which is 1.8 pH units above the theoretical isoelectric pH of the protein (calculated on the basis of considerations of the amino acid sequence data for the mature protein (data not given)). At this pH, the HPI protein is expected to be only slightly negatively charged with a smaller potential drop occurring inside the pores, as opposed to the previous measurements undertaken at pH 8.6 where the protein has the maximum amount of negative charge.14 Although the impedimetric response of the HPI S-layer to Ca+2 ions is suppressed in buffer solutions, we used a 10 mM buffer to permit detection. Significantly, the spectra and the calculated capacitance values indicate a further decrease in the response (sensitivity 0.24 pF‚M-1). The vertex-pore transport mechanism described here shares similarities with that of analogous transmembrane biological pores.29 Similar results have been reported by others for synthetic nanopores, wherein both experimental evidence and theoretical evidence point to the substantial role of surface currents in mediating nanoscale ionic transport. Surface currents have also been found to be responsible for cationic selectivity, since the barriers to ion transport are mainly electrostatic in nature. Other factors potentially affecting pore selectivity include the hydrophobicity of the pore and of the ions being transported, as well as the hydration energy of the ions.30 Indeed, the latter two factors would seem to account for the observed selectivity of the S-layer vertex regions to Ca2+ versus Mg2+. Although these two particular ions have the same charge, the ionic radius
of Ca2+ (0.99 Å) is larger than that for Mg2+ (0.65 Å); therefore, if size is the major selectivity factor at play, then a higher sensitivity should have been observed for Mg2+ instead of Ca2+. On the other hand, Ca2+ has a higher lipophilicity and lower enthalpy of hydration; such properties apparently allow the ions to lose their hydration shell and to more easily traverse the vertex regions of the S-layer. Siwy et al.25 have recently investigated calcium-induced voltage gating in synthetic conical nanopores. Their results also point to transient binding of calcium inside the pore walls and to differential behavior of the pores in the presence of manganese ions. Conclusions. We have monitored the ionic interactions of the D. radiodurans S-layer (HPI) protein with different species of cations and anions using electrochemical impedance spectroscopy. A strong interaction with calcium ions was established and is attributed to the penetration of the calcium ions into the Helmholtz plane of the double layer, where specific adsorption of ions to the S-layer occurs. Closer examination of the phenomenon led to strong indications that the calcium ions are being transported through the vertex regions of the HPI protein lattice. Ion transport appears to be mainly due to electrical gradients, presumed here to originate from the negative charges found on the surface of the protein. To the best of our knowledge, this is the first detailed investigation of the ion-gating properties of microbial S-layer protein arrays. Bioelectronic devices with analogous properties have been previously described for ion channels such as the R-hemolysin pore; however, in those cases there is a need for a support matrix for the pore, i.e., a bilayer lipid membrane. This not only introduces extra steps in the fabrication procedure, but also significantly compromises the stability of the device, since bilayer lipid membranes are quite fragile. The S-layerbased device, on the other hand, is a stand-alone device that can be easily fabricated and is extremely robust and stable.
Nanoporous S-Layer Protein Lattices Measurements carried out even 3 months after device fabrication showed no change in the performance characteristics. Furthermore, the S-layer system allows for great versatility, since the use of different S-layers with different lattice constants can potentially lead to the rapid production of large numbers of sensing devices with different performance characteristics. It is anticipated that further optimization of the surface immobilization and protein self-assembly procedures will enable better control over the number of S-layer monomers employed per device and thus allow us to obtain a quantitative estimate of the current and actual number of ions flowing through each pore, which is currently not possible. Moreover, the fabrication of large-area, free-standing S-layer membranes may ultimately permit the precise determination of the intrinsic through-vertex diffusion rate. Beyond having implications in the general understanding of the mechanisms occurring in ion-channel functions, the data reported here provide new insights into the possible physiological role of S-layers. Currently, significant debate exists regarding the permeability of the pore-vertex regions of such proteins. This is the first time that an ion-gating functionality has been shown to take place through the vertex regions of the HPI S-layer. Acknowledgment. We thank J. Scott Bunch (Laboratory of Atomic and Solid State Physics, Cornell University) for assistance with the AFM imaging of S-layers using carbon nanotube probe tips. This work was supported in part by the Cornell Nanobiotechnology Center (NBTC), an STC Program of the National Science Foundation under Agreement No. ECS9876771. We also acknowledge additional funding support from the National Science Foundation under a Nanoscale Interdisciplinary Research Team (NIRT) grant (NSF-0403990). Supporting Information Available: Schematic of the electrochemical setup (Figure S1), detailed control experiments of the impedance spectra of bare DDS-modified Si and HPImodified Si with all ionic species (Figures S2-S4), and SEM images of the Au NPs adsorbed onto the HPI S-layer (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.
J. Phys. Chem. C, Vol. 111, No. 35, 2007 13237 References and Notes (1) Bayley, H.; Martin, C. R. Chem. ReV. (Washington, D.C.) 2000, 100, 2575. (2) Pajkossy, T. Solid State Ionics 1997, 94, 123. (3) Bayley, H. Nat. Chem. Biol. 2006, 2, 11. (4) Schmidt, J. J. Mater. Chem. 2005, 15, 831. (5) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature (London) 1997, 387, 580. (6) Mara, A.; Siwy, Z.; Trautmann, C.; Wan, J.; Kamme, F. Nano Lett. 2004, 4, 497. (7) Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer: Sunderland, MA, USA, 2001. (8) Heins, E. A.; Siwy, Z. S.; Baker, L. A.; Martin, C. R. Nano Lett. 2005, 5, 1824. (9) Kasianowicz, J. J.; Henrickson, S. E.; Weetall, H. H.; Robertson, B. Anal. Chem. 2001, 73, 2268. (10) Howorka, S.; Cheley, S.; Bayley, H. Nat. Biotechnol. 2001, 19, 636. (11) Holden, M. A.; Bayley, H. J. Am. Chem. Soc. 2005, 127, 6502. (12) Smeets, R. M. M.; Keyser, U. F.; Krapf, D.; Wu, M.-Y.; Dekker, N. H.; Dekker, C. Nano Lett. 2006, 6, 89. (13) Keyser, U. F.; Koeleman, B. N.; Van Dorp, S.; Krapf, D.; Smeets, R. M. M.; Lemay, S. G.; Dekker, N. H.; Dekker, C. Nat. Phys. 2006, 2, 473. (14) Sleytr, U. B.; Messner, P.; Pum, D.; Sara, M. Angew. Chem., Int. Ed. 1999, 38, 1034. (15) Bergkvist, M.; Mark, S. S.; Yang, X.; Angert, E. R.; Batt, C. A. J. Phys. Chem. B 2004, 108, 8241. (16) Mark, S. S.; Bergkvist, M.; Yang, X.; Teixeira, L. M.; Bhatnagar, P.; Angert, E. R.; Batt, C. A. Langmuir 2006, 22, 3763. (17) Schuster, B.; Pum, D.; Sara, M.; Braha, O.; Bayley, H.; Sleytr, U. B. Langmuir 2001, 17, 499. (18) Pum, D.; Sleytr, U. B. Trends Biotechnol. 1999, 17, 8. (19) Schuster, B.; Sleytr Uwe, B. Bioelectrochemistry (Amsterdam, Netherlands) 2002, 55, 5. (20) Weigert, S.; Sara, M. J. Membr. Sci. 1996, 121, 185. (21) Schmickler, W., Ed. Interfacial Electrochemistry; Oxford University Press: New York, 1996. (22) Stora, T.; Lakey, J. H.; Vogel, H. Angew. Chem., Int. Ed. 1999, 38, 389. (23) Barsoukov, E.; MacDonald, R. Impedance Spectroscopy: Theory, Experiment, and Applications, 2nd ed.; John Wiley & Sons: Chichester, U. K., 2005. (24) Park, S.-M.; Yoo, J.-S. Anal. Chem. 2003, 75, 455A. (25) Siwy, Z. S.; Powell, M. R.; Petrov, A.; Kalman, E.; Trautmann, C.; Eisenberg, R. S. Nano Lett. 2006, 6, 1729. (26) Offenhaeusser, A. Bioelectronics; Vol. 7, Willner, I., Katz, E., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2006. (27) Chaniotakis, N. A.; Alifragis, Y.; Konstantinidis, G.; Georgakilas, A. Anal. Chem. 2004, 76, 5552. (28) Siwy, Z.; Kosinska, I. D.; Fulinski, A.; Martin, C. R. Phys. ReV. Lett. 2005, 94, 048102/1. (29) Gu, L.-q.; Cheley, S.; Bayley, H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15498. (30) Beckstein, O.; Tai, K.; Sansom, M. S. P. J. Am. Chem. Soc. 2004, 126, 14694.
